Abstract Transcription is initially repressed during early embryo development and then globally activates in a processed called zygotic genome activation (ZGA). The precise timing of ZGA is critical for embryo development: delaying ZGA blocks gastrulation and cell differentiation; premature ZGA induction disrupts normal development. However, the nature of ZGA regulation and the identity of factors that control its onset have remained elusive. Three models have been proposed to explain the onset of ZGA: 1) embryos contain a timer or clock, 2) a sizer that measures cell volume, or 3) an event counter that tracks cell divisions. A timer could consist of maternal mRNAs encoding proteins, such as pluripotency factors, that are steadily translated after fertilization and whose accumulation induces ZGA. The sizer hypothesis supposes that when cells achieve a sufficiently small volume they reach a threshold DNA:cytoplasm ratio that overcomes a global block of zygotic transcription. Histones and DNA replication factors have been implicated in setting this block. The molecules that constitute a cell cycle counter are unknown. Time, cell size, and cell cycle number are intertwined, therefore it has been challenging to determine whether one or all of these parameters controls ZGA. Ultimately, our understanding of embryonic genome activation has been hampered by an inability to measure cell-to-cell variability in an embryo, a lack of genetic tools to deplete maternal factors implicated in ZGA regulation, and the challenge of altering cell or embryo dimensions. My lab recently developed a state-of-the-art technique to image ZGA in time and space at single-cell resolution in whole cleavage-stage embryos by labeling newly synthesized RNA with 5-ethynyl uridine (5-EU). Using this technique, we distinguish between ZGA regulatory mechanisms based exclusively on a timer, sizer or counter. In Aim 1, we will construct a spatial map of genome activation in single-cells of Xenopus and zebrafish blastula embryos. Additionally, by constricting embryo dimensions, we will generate mini-embryos to distinguish genome activation that is initiated by a cell cycle counter from or a cell volume sensor. In Aim 2, we will characterize the mechanisms by which core histones regulate ZGA onset, using a stem loop binding protein 2 mutant zebrafish embryos that contain significantly reduced levels of core histones. In Aim 3, we will determine whether translation of pluripotency factors constitutes a timer for triggering ZGA. The premise of this application is that a single model cannot explain the precise timing and patterning of ZGA. Specifically, we hypothesize that cells must reach a threshold size to relieve histone-based repression, and contain a sufficient level of pluripotency factors to induce zygotic transcription. The research proposed here will provide a new mechanistic understanding of embryonic genome activation ? a universal feature of developmental in all vertebrates, including humans - and how specific combinations of regulatory paradigms dictate patterning and timing of zygotic transcription.